Abstract Book 2010 - CIMT Annual Meeting

068 Guthmann | New targets & new leads
Cellular and humoral immune response to N-glycolyl-GM3
elicited by racotumomab, an anti-idiotypic vaccine
Marcelo D Guthmann 1 , Cecilia Venier 1 , Estrella Levy 1 , Mónica A Castro 2 , Gabriela Cinat 2 ,
Roberto Gómez 3 , Ana María Vázquez 4 , Leonardo Fainboim 1
1 Hospital de Clínicas José de San Martín, University of Buenos Aires, Argentina
2 Instituto de Oncología A. Roffo, University of Buenos Aires, Argentina
3 Laboratorio Elea, Buenos Aires, Argentina
4 Center of Molecular Immunology, Havana, Cuba
Gangliosides are a family of sialylated glycolipids
which are normal components of the cell membrane.
They have been found to be important actors in
multiple aspects of cellular interaction with its environment
and with transmembrane signaling. As
such, they are involved in cancer progression and
have become the focus of several immunotherapeutic
approaches. Not all gangliosides are equally
immunogenic. N-acetyl-GM3, the main gangliosides
on the cell surface and the most abundant
ganglioside in normal serum, is one of the most
immunologically tolerated member of the family.
In contrast, N-glycolyl-GM3, is not expressed in
human normal tissues due to a species-specific
genetic mutation that abrogates the biosynthesis of
N-glycolylneuraminic acid (Neu5Gc). Neu5Gc has
been reported, however, in human tumors, and its
presence might be derived from dietary sources
or a yet unknown alternate synthesis pathway. Nglycolyl-GM3
is expressed in melanoma, breast and
lung cancer cells, and is highly immunogenic. As a
result, it has been considered as a target of choice
for immunotherapy.
We investigated with an extended vaccination protocol
the immunogenicity and toxicity profile of racotumomab,
an anti-idiotypic vaccine mimicking
N-glycolyl-GM3. The year-long vaccination scheme
consisted of six bi-weekly intradermal injections
followed by 10 monthly boosters. Nineteen patients
with high risk (stage III) or metastatic breast
cancer were vaccinated with different dose levels
of racotumomab (0.5, 1 and 2 mg). The humoral
and cellular responses to racotumomab and to the
targeted ganglioside were assessed at baseline and
throughout the treatment. Anti-idiotype antibodies
and anti-N-glycolyl-GM3 IgM and IgG antibodies
were determined by ELISA. Serum antibody reactivity
was also tested against P3X63 Ag8 653 murine
myeloma cells and B16 murine melanoma cells.
Frequency of N-glycolyl-GM3—reactive cells was
calculated by IFNγ ELISPOT. For that purpose, cryopreserved
PBMC and autologous DC were incubated
with N-glycolyl-GM3—containing liposomes.
Equivalent amounts of unloaded liposomes were
added to control wells. After a 24-h culture, cells in
each well were resuspended and transferred to triplicate
wells in an IFNγ-precoated ELISPOT plate.
Subsequent steps for detection of IFNγ-secreting
cells followed standard ELISPOT procedures. Patients
with no response at baseline and with a posttreatment
two-fold increase in the number of spots
in N-glycolyl-GM3 stimulated wells (versus unstimulated
wells) were considered as responsive.
All patients showed a strong antibody response to
N-glycolyl GM3. In addition, ganglioside-specific
IFNγ responses were recorded in 5 of 13 evaluable
patients.
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